This project is focused on the node of Ranvier of myelinated nerve. The general goal is to extend our work on establishing a better understanding of how the node functions and to continue to identify the locations of macromolecules in the nodal complex that are important to conduction of the nerve impulse and development Specific aim I is to determine the sources, mechanisms and roles of[Ca2+]i transients and the sites of voltage transients in the nodal complex. The ultimate objectives of these studies are twofold: to gain a better understanding of (1) the roles of paranodal glia in the regulation of the nodal ionic milieu; and (2) the mechanisms underlying alterations in threshold and changes in conduction velocity that occur following impulse activity. Experiments are to be conducted using optical methods for recording of voltage and (Ca2+]i transients as well as time-lapse Nomarski imaging. In these experiments we will use optical methods to determine the structural and physiological effects of drugs known to alter specific ion conductances. Specific aim II involves studies of 5-HT receptors in Schwann cells. The objectives are to determine the type of 3-HT receptor responsible for Schwann cell [Ca2+]i transients in vitro and determine if these receptors are present in vivo. Specific aim IIl is to determine the sites of previously characterized and newly identified macromolecules in the node of Ranvier complex using immunolocalization techniques as well as to determine the sequence of appearance of macromolecules during development, regeneration and remyelination. The objectives of these studies are (1) to identify the precise locations of membrane proteins and other macromolecular constituents that we believe are important to nodal function and development; and (2) to study the developmental sequence for the appearance of the subset of these macromolecules that may be involved in defining the initial locations of nodes along premyelinated or remyelinating fibers. Our first experiments localizing macromolecules in the nodal complex win follow up on interesting preliminary results with newly characterized antibodies to K+ channels, ryanodine receptors, Na+/Ca2+ exchangers and H2O channels, as well as Na+/K+ ATPases, tyrosine kinases and proteins phosphorylated on tyrosine. Simultaneously we will conduct experiments aimed at gaining new insight into the initial stages of nodal development, remyelination and regeneration, using antibodies specific for the voltage dependent Na+ and K+ channels. In these experiments the aim is to determine the sequence of development of macromolecular components of the nodal complex. A special interest will be to determine if one of the constituents disclosed during the antibody localization studies appears prior to the arrival of myelinating cells thus participating in the early definition of the node of Ranvier. The results of these studies will provide a better understanding of the relationship between structure and function at the node of Ranvier and the underlying macromolecular basis for impaired conduction in demyelinating disease.